In the past granting period we made a paradigm shifting discovery in which we showed that the two key virulence factors from Bacillus anthracis, edema factor (EF) and lethal factor (LF) act cooperatively to inhibit protein trafficking to cell-cll junctions. EF (a highly active adenylate cyclase) reduces the levels and activity of a small GTPase (Rab11) required in the final step of endocytic recycling to the cell surface, while LF (a metalloprotease that cleaves and inactivates MEKs) inhibits the Rab11 binding partner, Sec15, a component of the exocyst complex located at the plasma membrane. By inhibiting endocytic recycling, anthrax toxins also reduce levels of cell adhesion molecules (e.g., cadherins) and signaling proteins (e.g., Notch components) at cell-cell junctions, resulting in barrier disruption We discovered this novel effect of anthrax toxins using the model genetic system Drosophila melanogaster (fruit fly) and then validated these effects in human vascular endothelial cells as well as in vivo in mice. Another key finding, with important practical implications, was that over-expression of Rab11, can reverse the junction disrupting effects of EF in both flies and human cells. In the current grant, we propose two integrated aims to determine the mechanisms by which anthrax toxins inhibit endocytic recycling (Aim 1), and to investigate the role of endocytic recycling the pathogenesis of anthrax infection (Aim 2). In Aim 1.1, we will focus on how high levels of cAMP produced by EF interfere with effector pathways promoting exocyst function and lead to reduced Rab11 protein levels. In Aim 1.2, we will determine whether inhibiting MEKs can account for the ability of LF to block exocyst-mediated trafficking, and whether exocyst function is regulated by down-stream MAP-Kinases. In Aim 2.1, we will first survey the effects of anthrax toxins on expression of Rab11 and Sec15 in known target organs and cell types. We will also examine how various cell biological processes and barrier-regulating pathways contribute to the permeabilizing effects of anthrax toxins in vascular endothelial cells since vascular collapse is a frequent cause of death in anthrax. Finally, in Aim 2.2, we will determine whether increasing Rab11 levels or treating with known barrier protective agents can reverse the vascular leakage caused by EF. The proposed studies are highly relevant to treating anthrax since toxins typically reach critical lethal levels in the blood just as patients begin to seek medical intervention, when antibiotics are no longer effective. Thus, treatments based on restoring endocytic recycling could be used in conjunction with existing anti-toxin therapies (e.g., anti-toxin antibodies, enzymatic inhibitors) to neutralize toxins already present in the circulation. An advantage of barrier-protective agents is that they would intervene at the very last step when vascular integrity collapses and other organ systems fail. Such barrier enhancing compounds could also have broad applications to a variety of barrier disruptive diseases including other infectious diseases, cardiac ischemia, asthma, dermatitis, inflammatory bowel diseases, cancer, ciliary diseases, and neurodegenerative disorders.